This invention relates to therapeutic modalities for treatment of neoplastic disease. More specifically, this invention involves synthetic peptides that selectively destroy malignant and transformed cells, and a method for treatment of neoplastic disease based thereon. The present invention also relates to viral therapy for cancer.
The p53 protein is a vital regulator of the cell cycle. It blocks the oncogenic effects of a number of oncogene proteins that induce mitosis, in part by blocking transcription of proteins that induce mitosis and by inducing the transcription of proteins that block mitosis, and promote apoptosis. Absence of the p53 protein is associated with cell transformation and malignant disease. Haffner, R & Oren, M. (1995) Curr. Opin. Genet. Dev. 5: 84-90.
The p53 protein molecule consists of 393 amino acids. It includes domains that bind to specific sequences of DNA in a DNA-binding domain that consists of residues 93-313. The crystal structure of this region has been determined by x-ray crystallography. Residues 312-393 are involved in the formation of homotetramers of the p53 protein. Residues 1-93 are involved in regulation of the activity and half life of the p53 protein.
The p53 protein binds to another important regulatory protein, the MDM-2 protein. The MDM-gene that encodes the MDM-2 protein is a known oncogene. The MDM-2 protein forms a complex with the p53 protein, which results in the degradation of the p53 protein by a ubiquination pathway. The p53 protein binds to MDM-2 protein using an amino acid sequence that includes residues 14-22 of the p53 protein, which are invariant. The entire MDM-2 protein binding domain of the p53 protein spans residues 12-26. Haffner, R & Oren, M. (1995) Curr. Opin. Genet. Dev. 5: 84-90.
Considering that the MDM-2 protein is the expression product of a known oncogene, it is not surprising that MDM-2 protein is a very important regulatory protein. Moreover, overexpression or amplification of MDM-2 protein has been found in 40-60% of human malignancies, including 50% of human breast tumors. It has been suggested that formation of a complex between the p53 protein and the MDM-2 protein may result in the inhibition of transcription activity of the p53 protein, and thus the anti-tumor effect of the molecule by blocking of an activation domain of the p53 protein, or of a DNA binding site within it. More generally, these and other experimental observations have been interpreted as suggesting that the anti-tumor effect of the p53 protein might be enhanced by peptides capable of interfering with the binding of the MDM-2 protein to the p53 protein. Indeed, a number of investigators have suggested that the MDM-2/p53 complex might be a target for rational drug design. See, e.g., Christine Wasylyk et al., “p53 Mediated Death of Cells Overexpressing MDM2 by an Inhibitor of MDM2 Interaction with p53”, Oncogene, 18, 1921-34 (1999), and U.S. Pat. No. 5,770,377 to Picksley et al.
The ability of the adenovirus to infect a broad spectrum of cells and its high infection efficiency has made it a prominent candidate for cancer therapy (Russell, 2000). The adenovirus consists of an icosahedral capsid. The capsid, which encloses the viral genome, is composed of three major proteins: a hexon, a penton base, and a protein called knobbed fibre. In addition, several minor proteins, VI, VIII, IX, IIIa and IVa2 and a virus-encoded protease are also present. The viral genome is a linear, double-stranded DNA with a terminal protein attached covalently to the 5′ termini, which have long terminal repeats (LTRs). The DNA is also associated with protein VII and a peptide known as mu (Russell, 2000). The adenoviral genome can be subdivided into three functional categories: the early genes, which encode for the E1A, E1B, E2, E3 and E4 proteins, the delayed genes and the single major late unit. DNA replication, viral gene transcription, host cell immune suppression and inhibition of host cell apoptosis can be attributed to the early gene products. The products of the late genes are required for virus assembly (Wang et al., 2000).
The infectious cycle of the Adenovirus can be defined in an early phase and a late phase. The early phase covers the entry of the virus into the host cell, followed by the passage of the viral genome into the nucleus and selective transcription and translation of the early genes Russell, 2000). In these early events the virus takes over the functions of the host cell in order to facilitate the replication of the viral DNA, which leads to the translation of the late genes. A key factor in cell tropism is receptor recognition. The protein knobbed fibre binds with high affinity to a cell surface receptor known as Coxsackie Adenovirus Receptor (CAR). Infection of cells is first initiated by the binding of the knob fibre to CAR. Internalization of the virus involves the additional binding of the penton base to surface integrins—proteins which are involved with the extracellular matrix in cell adhesion, cell-cell junctions and other cell-cell related phenomena (Mathias et al., 1998; Meredith et al., 1996). A number of signaling pathways are then induced that facilitate the clathrin-mediated endocytosis of the viral particle. Endocytosis involves the invagination of the viral particle followed by the pinching off the plasma membrane resulting in the development of endocytic vesicles. The virus-encoded protease then disrupts the viral capsid by degradation of the structural protein VI. The virus is then transported into the nuclear membrane, where the genome enters into the nucleus leading to initial transcription. It is believed that the viral genome is able to access the host nucleus through the help of the cellular protein p32. p32 is found primarily in the mitochrondria, but is also present in the nucleus. The protein acts as an intracellular shuttle between the mitochondria and nucleus. It appears that the virus is able to take over this intracellular shuttle system in order to gain access to the nucleus (Russell, 2000).
Low morbidity and high-level transgene expression have made adenovirus a very attractive vector in functional studies. However, the supremacy of the immune response in vivo has been a limiting factor in the practical development of vectors. The duration of the transgene expression is limited in vivo to a large extent by the host's anti-adenoviral immune response toward the infected cell (Yang et al., 1996). Epithelial cells for example are able to release antimicrobial peptides that inhibit adenoviral infection (Gropp et al. 1999). Other defense mechanisms include the release of cellular proteins known as interferons, upon early infection. Interferons activate the cellular Jak/STAT pathway; which results in the activation of Interferon-response elements that regulate the transcription of a range of gene products that can eliminate the infected cells and protect healthy cells from infection (Goodbourn et al., 2000). T-cell recognition of viral antigens presented on the surface of the infected cells in context of MHC Class I antigens can result in the transfer of perforin from cytotoxic T-cells and the lysis of infected cells. Viral activity can also invoke apoptotic pathways leading to programmed cell death. Particularly in cancer therapy, where the goal is to eliminate all transformed cells, the activation of the immune response is a desired effect.
The tumor suppressor p53 plays a primary role in apoptosis and cell cycle arrest (Alberts et al., 2002). This protein regulates the transcription of anti-proliferative proteins such as Bax. Bax stimulates the release of cytochrome c from mitochondria, which binds to the adaptor protein Apaf-1 and the whole complex then activates procaspases leading to cell death (Alberts et al., 2002). p53 is regulated by the oncoprotein murine double minute 2, mdm2. Mdm2 is able to form complexes with p53 protein leading to its ubiquitination and its degradation by proteasomes (Uchida et al., 2002). p53 provides protection from dangerous events such as uncontrolled cell proliferation and mutations that may arise in the cell. A defective p53 gene can have numerous adverse consequences for the cell. Cells defective in p53 or containing mutated p53 are able to circumvent apoptosis and continue their proliferation. DNA damages within these cells accumulate with each new division, while the cell is unable to repair them. Thus, loss of p53 function is closely related to the development of cancer. Loss of p53 function allows mutant cells to continue through the cell cycle, to bypass apoptosis and the accumulation of cancer-promoting mutations as they divide (Alberts et al., 2002).
Mutations in the p53 gene and its relation to the development of a wide variety of human tumors have prompted the development of vectors incorporating wild-type p53. Such vectors have been tested successfully in vitro and in vivo in anaplastic thyroid cancer, human malignant gliomas and breast cancer with success (Russell, 2000).
In addition to providing peptides which selectively kill malignant or transformed cells, the present invention provides adenoviral vectors (AdV) that incorporate a transgene, which codes for such peptides. Administration of such vectors to malignant and transformed cells results in death of such cells. The subject AdV expression vehicles are especially useful for their anti-proliferative properties in treating different neoplastic diseases including pancreatic cancer.